Methods for array preparation using substrate rotation

ABSTRACT

Methods are provided for the preparation of polymer arrays on wafers wherein the wafers are rotated between synthesis steps to provide more uniform results across the entire wafer.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of 60/209,411, filed Jun. 1, 2000,the disclosure of which is incorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable

BACKGROUND OF THE INVENTION

The present invention relates to improved methods for preparingsupport-bound nucleic acid arrays. More particularly, the inventionrelates to methods of preparing the arrays wherein a planar arraysubstrate is held in a substantially vertical position and rotatedduring the course of nucleic acid synthesis.

Substrate-bound nucleic acid arrays, such as the Affymetrix DNA Chip,enable one to test hybridization of a target nucleic acid molecule tomany thousands of differently sequenced nucleic acid probes at featuredensities greater than about five hundred per 1 cm². Becausehybridization between two nucleic acids is a function of theirsequences, analysis of the pattern of hybridization provides informationabout the sequence of the target molecule. The technology is useful forde novo sequencing and re-sequencing of nucleic acid molecules and alsohas important diagnostic uses in discriminating genetic variants thatmay differ in sequence by one or a few nucleotides. For example,substrate-bound nucleic acid arrays are useful for identifying geneticvariants of infectious diseases, such as HIV, or genetic diseases, suchas cystic fibrosis.

In one version of the substrate-bound nucleic acid array, the targetnucleic acid is labeled with a detectable marker, such as a fluorescentmolecule. Hybridization between a target and a probe is determined bydetecting the fluorescent signal at the various locations on thesubstrate. The amount of signal is a function of the thermal stabilityof the hybrids. The thermal stability is, in turn, a function of thesequences of the target-probe pair: AT-rich regions of DNA melt at lowertemperatures than GC-rich regions of DNA. This differential in thermalstabilities is the primary determinant of the breadth of DNA meltingtransitions, even for nucleic acids.

Depending upon the length of the nucleic acid probes, the number ofdifferent probes on a substrate, the length of the target nucleic acid,and the degree of hybridization between sequences containing mismatches,among other things, a hybridization assay carried out on asubstrate-bound nucleic acid array can generate thousands of data pointsof different signal strengths that reflect the sequences of the probesto which the target nucleic acid hybridized. This information canrequire a computer for efficient analysis. The fact of differentialfluorescent signal due to differences in thermal stability of hybridscomplicates the analysis of hybridization results, especially fromcombinatorial nucleic acid arrays for de novo sequencing and customnucleic acid arrays for specific re-sequencing applications.Modifications in custom array designs have contributed to simplifyingthis problem.

Further complications can arise and lead to variability in diagnostic orsequencing results. For example, degradation of nucleic acid probes,either during the synthesis steps or on standing can lead to variabilityin assay results. Accordingly, there exists a need for additionalmethods of nucleic acid array preparation, and the arrays themselves, toprovide more robust tools for the skilled researcher. The presentinvention provides such methods and arrays.

SUMMARY OF THE INVENTION

The present invention provides an improved method for forming nucleicacid arrays, or more generally, any oligomeric arrays. In a number ofarray fabrication technologies, the substrate on which synthesis takesplace is held in a substantially vertical position. Once regions of thesubstrate have been activated, a suitable monomer (typically insolution) is contacted with the substrate for attachment to the nascentoligomer. Underlying the present invention was the discovery that lowerportions of arrays, in some instances, yielded suboptimal products.Accordingly, in a fabrication process on a substrate or wafer dividedinto 49 chips (a 7×7 chip), there was a large intra wafer variationbetween the top and bottom probe arrays. Surprisingly, by rotating thesubstrate (or wafer) during array synthesis, essentially all chipsproduced passed the stringent quality control criteria.

In view of the above, the present invention provides a method ofpreparing a nucleic acid array on a support, wherein each nucleic acidoccupies a separate known region of the support, said synthesizingcomprising:

(a) activating a region of the support;

(b) attaching a nucleotide to a first region, said nucleotide having amasked reactive site linked to a protecting group;

(c) repeating steps (a) and (b) on other regions of said support wherebyeach of said other regions has bound thereto another nucleotidecomprising a masked reactive site link to a protecting group, whereinsaid another nucleotide may be the same or different from that used instep (b);

(d) removing the protecting group from one of the nucleotides bound toone of the regions of the support to provide a region bearing anucleotide having an unmasked reactive site;

(e) binding an additional nucleotide to the nucleotide with an unmaskedreactive site;

(f) repeating steps (d) and (e) on regions of the support until adesired plurality of nucleic acids is synthesized, each nucleic acidoccupying separate known regions of the support;

wherein the surface of said substrate is maintained in a position whichis vertical or within about 30 degrees of vertical during the attachingand binding steps, and

wherein the substrate is rotated around an axis perpendicular to saidsurface by an amount of from about 20 degrees to about 180 degrees, saidrotating being done prior to, coincident with or subsequent to at leastone of said attaching or binding steps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the rotation positions for a wafer during arraypreparation.

FIG. 2 is a graph that illustrates the average difference in controlversus rotated arrays.

FIG. 3 is a graph that illustrates the average differences for a controlwafer (296) versus the rotated wafers (297 and 299).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Definitions

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

“Nucleic acid library” or “array” is an intentionally created collectionof nucleic acids which can be prepared either synthetically orbiosynthetically and screened for biological activity in a variety ofdifferent formats (e.g., libraries of soluble molecules; and librariesof oligos tethered to resin beads, silica chips, or other solidsupports). Additionally, the term “array” is meant to include thoselibraries of nucleic acids which can be prepared by spotting nucleicacids of essentially any length (e.g., from 1 to about 1000 nucleotidemonomers in length) onto a substrate. The term “nucleic acid” as usedherein refers to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides, that comprise purine andpyrimidine bases, or other natural, chemically or biochemicallymodified, non-natural, or derivatized nucleotide bases. The backbone ofthe polynucleotide can comprise sugars and phosphate groups, as maytypically be found in RNA or DNA, or modified or substituted sugar orphosphate groups A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs. The sequence ofnucleotides may be interrupted by non-nucleotide components. Thus theterms nucleoside, nucleotide, deoxynucleoside and deoxynucleotidegenerally include analogs such as those described herein. These analogsare those molecules having some structural features in common with anaturally occurring nucleoside or nucleotide such that when incorporatedinto a nucleic acid or oligonucleoside sequence, they allowhybridization with a naturally occurring nucleic acid sequence insolution. Typically, these analogs are derived from naturally occurringnucleosides and nucleotides by replacing and/or modifying the base, theribose or the phosphodiester moiety. The changes can be tailor made tostabilize or destabilize hybrid formation or enhance the specificity ofhybridization with a complementary nucleic acid sequence as desired.

“Solid support”, “support”, and “substrate” are used interchangeably andrefer to a material or group of materials having a rigid or semi-rigidsurface or surfaces. In many embodiments, at least one surface of thesolid support will be substantially flat, although in some embodimentsit may be desirable to physically separate synthesis regions fordifferent compounds with, for example, wells, raised regions, pins,etched trenches, or the like. According to other embodiments, the solidsupport(s) will take the form of beads, resins, gels, microspheres, orother geometric configurations.

“Predefined region” or “preselected region” refers to a localized areaon a solid support which is, was, or is intended to be used forformation of a selected molecule and is otherwise referred to herein inthe alternative as a “selected” region, a “known” region, or a “known”location. The predefined or known region may have any convenient shape,e.g., circular, rectangular, elliptical, wedge-shaped, etc. For the sakeof brevity herein, “predefined regions” are sometimes referred to simplyas “regions.” In some embodiments, a predefined or known region and,therefore, the area upon which each distinct compound is synthesized issmaller than about 1 cm² or less than 1 mm². Within these regions, themolecule synthesized therein is preferably synthesized in asubstantially pure form. In additional embodiments, a known region canbe achieved by physically separating the regions (i.e., beads, resins,gels, etc.) into wells, trays, etc. Accordingly, materials (e.g.,nucleic acids) can be synthesized or attached to any particular regionby any known methods or means.

General

Nucleic acid arrays having single-stranded nucleic acid probes havebecome powerful research tools for identifying and sequencing new genes.Other arrays of unimolecular double-stranded DNA have been developedwhich are useful in a variety of screening assays and diagnosticapplications (see, for example, U.S. Pat. No. 5,556,752). Still otherarrays have been described in which a ligand or probe (a peptide, forexample), is held in a conformationally restricted position by twocomplementary nucleic acids, at least one of which is attached to asupport. Common to each of these types of arrays is the presence of asupport-bound nucleic acid and the exquisite sensitivity exhibited bythe arrays. Unfortunately, the sensitivity of these arrays can becompromised if the nucleic acids are degraded or are not prepared insufficient quantity on the support.

In order to provide the researcher with arrays of uncompromising qualityand reproducible performance, arrays should be prepared using high yieldreactions and excluding any component which could negatively impactsynthesis yield or the performance of the array.

The present invention derives from the discovery that improved yieldscan be obtained if nucleic acid array substrates are held in asubstantially vertical position and rotated during and/or betweensynthesis steps of nucleic acid array preparation.

Embodiments of the Invention

In view of the above discoveries, the present invention provides animproved method of preparing a nucleic acid array on a support. In ageneral sense, the method comprises synthesizing a plurality of nucleicacids on a support wherein the support is held in a substantiallyvertical position and rotated about an axis perpendicular to thesubstrate surface during the course of synthesis.

Synthesis of Nucleic Acid Arrays

In the present invention, nucleic acid arrays can be prepared using avariety of synthesis techniques directed to high-density arrays ofnucleic acids on solid supports. In brief, the methods can includelight-directed methods, flow channel or spotting methods, pin-basedmethods, or combinations thereof. For light-directed methods, see, forexample, U.S. Pat. Nos. 5,143,854, 5,424,186 and 5,510,270. Fortechniques using mechanical methods, see PCT No. 92/10183, U.S. Pat. No.5,384,261 and PCT/US99/00730. For a description of pin-based methods,see U.S. Pat. No. 5,288,514. A brief description of these methods isprovided below. The methods of the present invention are equallyamenable to the preparation of unimolecular double-stranded DNA arrays(see U.S. Pat. No. 5,556,752). In addition, the nucleic acid arraysprepared in the present methods will also include those arrays in whichindividual nucleic acids are interrupted by non-nucleotide portions(see, for example U.S. Pat. No. 5,556,752 in which probes such aspolypeptides are held in a conformationally restricted manner bycomplementary nucleic acid fragments).

Various additional techniques for large scale polymer synthesis areknown. Some examples include the U.S. Pat. Nos. 5,143,854, 5,242,979,5,252,743, 5,324,663, 5,384,261, 5,405,783, 5,412,087, 5,424,186,5,445,934, 5,451,683, 5,482,867, 5,489,678, 5,491,074, 5,510,270,5,527,681, 5,550,215, 5,571,639, 5,593,839, 5,599,695, 5,624,711,5,631,734, 5,677,195, 5,744,101, 5,744,305, 5,753,788, 5,770,456,5,831,070, and 5,856,011, all of which are incorporated by referenceherein.

Libraries on a Single Substrate

Light-Directed Methods

For those embodiments using a single solid support, the nucleic acids ofthe present invention can be formed using techniques known to thoseskilled in the art of polymer synthesis on solid supports. Preferredmethods include, for example, “light directed” methods which are onetechnique in a family of methods known as VLSIPS™ methods. The lightdirected methods discussed in U.S. Pat. No. 5,143,854 involve activatingknown regions of a substrate or solid support and then contacting thesubstrate with a preselected monomer solution. The known regions can beactivated with a light source, typically shown through a mask (much inthe manner of photolithography techniques used in integrated circuitfabrication). Other regions of the substrate remain inactive becausethey are blocked by the mask from illumination and remain chemicallyprotected. Thus, a light pattern defines which regions of the substratereact with a given monomer. By repeatedly activating different sets ofknown regions and contacting different monomer solutions with thesubstrate, a diverse array of nucleic acids is produced on thesubstrate. Of course, other steps such as washing unreacted monomersolution from the substrate can be used as necessary.

The VLSIPS™ methods are preferred for the methods described herein.Additionally, the surface of a solid support, optionally modified withspacers having photolabile protecting groups such as NVOC and MeNPOC, isilluminated through a photolithographic mask, yielding reactive groups(typically hydroxyl groups) in the illuminated regions. A3′-O-phosphoramidite activated deoxynucleoside (protected at the5′-hydroxyl with a photolabile protecting group) is then presented tothe surface and chemical coupling occurs at sites that were exposed tolight. Following capping, and oxidation, the substrate is rinsed and thesurface illuminated through a second mask, to expose additional hydroxylgroups for coupling. A second 5′-protected, 3′-O-phosphoramiditeactivated deoxynucleoside is presented to the surface. The selectivephotodeprotection and coupling cycles are repeated until the desired setof nucleic acids is produced. Alternatively, an oligomer of from, forexample, 4 to 30 nucleotides can be added to each of the known regionsrather than synthesize each member in a monomer by monomer approach.Methods for light-directed synthesis of DNA arrays on glass substratesare also described in McGall et al., J. Am. Chem. Soc., 119:5081-5090(1997).

For the above light-directed methods wherein photolabile protectinggroups and photolithography are used to create spatially addressableparallel chemical synthesis of a nucleic acid array (see also U.S. Pat.No. 5,527,681), computer tools may be used to assist in forming thearrays. For example, a computer system may be used to select nucleicacid or other polymer probes on the substrate, and design the layout ofthe array as described in, for example, U.S. Pat. No. 5,571,639.

Flow Channel or Spotting Methods

Additional methods applicable to library synthesis on a single substrateare described in U.S. Pat. No. 5,384,261 and in PCT/US99/00730. In themethods disclosed in this patent and PCT publication, reagents aredelivered to the substrate by either (1) flowing within a channeldefined on known regions or (2) “spotting” on known regions. However,other approaches, as well as combinations of spotting and flowing, maybe employed. In each instance, certain activated regions of thesubstrate are mechanically separated from other regions when the monomersolutions are delivered to the various reaction sites.

A typical “flow channel” method applied to the compounds and librariesof the present invention can generally be described as follows. Diversenucleic acid sequences are synthesized at selected regions of asubstrate or solid support by forming flow channels on a surface of thesubstrate through which appropriate reagents flow or in whichappropriate reagents are placed. For example, assume a monomer “A” is tobe bound to the substrate in a first group of selected regions. Ifnecessary, all or part of the surface of the substrate in all or a partof the selected regions is activated for binding by, for example,flowing appropriate reagents through all or some of the channels, or bywashing the entire substrate with appropriate reagents. After placementof a channel block on the surface of the substrate, a reagent having themonomer A flows through or is placed in all or some of the channel(s).The channels provide fluid contact to the first selected regions,thereby binding the monomer A on the substrate directly or indirectly(via a spacer) in the first selected regions.

Thereafter, a monomer B is coupled to second selected regions, some ofwhich may be included among the first selected regions. The secondselected regions will be in fluid contact with a second flow channel(s)through translation, rotation, or replacement of the channel block onthe surface of the substrate; through opening or closing a selectedvalve; or through deposition of a layer of chemical or photoresist. Ifnecessary, a step is performed for activating at least the secondregions. Thereafter, the monomer B is flowed through or placed in thesecond flow channel(s), binding monomer B at the second selectedlocations. In this particular example, the resulting sequences bound tothe substrate at this stage of processing will be, for example, A, B,and AB. The process is repeated to form a vast array of sequences ofdesired length at known locations on the substrate.

After the substrate is activated, monomer A can be flowed through someof the channels, monomer B can be flowed through other channels, amonomer C can be flowed through still other channels, etc. In thismanner, many or all of the reaction regions are reacted with a monomerbefore the channel block must be moved or the substrate must be washedand/or reactivated. By making use of many or all of the availablereaction regions simultaneously, the number of washing and activationsteps can be minimized.

One of skill in the art will recognize that there are alternativemethods of forming channels or otherwise protecting a portion of thesurface of the substrate. For example, according to some embodiments, aprotective coating such as a hydrophilic or hydrophobic coating(depending upon the nature of the solvent) is utilized over portions ofthe substrate to be protected, sometimes in combination with materialsthat facilitate wetting by the reactant solution in other regions. Inthis manner, the flowing solutions are further prevented from passingoutside of their designated flow paths.

The “spotting” methods of preparing nucleic acid libraries can beimplemented in much the same manner as the flow channel methods. Forexample, a monomer A can be delivered to and coupled with a first groupof reaction regions which have been appropriately activated. Thereafter,a monomer B can be delivered to and reacted with a second group ofactivated reaction regions. Unlike the flow channel embodimentsdescribed above, reactants are delivered by directly depositing (ratherthan flowing) relatively small quantities of them in selected regions.In some steps, of course, the entire substrate surface can be sprayed orotherwise coated with a solution. In preferred embodiments, a dispensermoves from region to region, depositing only as much monomer asnecessary at each stop. Typical dispensers include a micropipette todeliver the monomer solution to the substrate and a robotic system tocontrol the position of the micropipette with respect to the substrate,or an ink-jet printer. In other embodiments, the dispenser includes aseries of tubes, a manifold, an array of pipettes, or the like so thatvarious reagents can be delivered to the reaction regionssimultaneously. Still other spotting methods are described inPCT/US99/00730.

Pin-Based Methods

Another method which is useful for the preparation of nucleic acidarrays and libraries involves “pin based synthesis.” This method isdescribed in detail in U.S. Pat. No. 5,288,514. The method utilizes asubstrate having a plurality of pins or other extensions. The pins areeach inserted simultaneously into individual reagent containers in atray. In a common embodiment, an array of 96 pins/containers isutilized.

Each tray is filled with a particular reagent for coupling in aparticular chemical reaction on an individual pin. Accordingly, thetrays will often contain different reagents. Since the chemistrydisclosed herein has been established such that a relatively similar setof reaction conditions may be utilized to perform each of the reactions,it becomes possible to conduct multiple chemical coupling stepssimultaneously. In the first step of the process the invention providesfor the use of substrate(s) on which the chemical coupling steps areconducted. The substrate is optionally provided with a spacer havingactive sites. In the particular case of nucleic acids, for example, thespacer may be selected from a wide variety of molecules which can beused in organic environments associated with synthesis as well asaqueous environments associated with binding studies. Examples ofsuitable spacers are polyethyleneglycols, dicarboxylic acids, polyaminesand alkylenes, substituted with, for example, methoxy and ethoxy groups.Additionally, the spacers will have an active site on the distal end.The active sites are optionally protected initially by protectinggroups. Among a wide variety of protecting groups which are useful areFMOC, BOC, t-butyl esters, t-butyl ethers, and the like. Variousexemplary protecting groups are described in, for example, Atherton etal., SOLID PHASE PEPTIDE SYNTHESIS, IL Press (1989). In someembodiments, the spacer may provide for a cleavable function by way of,for example, exposure to acid or base.

Solid Supports

Solid supports used in the present invention include any of a variety offixed organizational support matrices. In some embodiments, the supportis substantially planar. In some embodiments, the support may bephysically separated into regions, for example, with trenches, grooves,wells and the like. Examples of supports include slides, beads and solidchips. Additionally, the solid supports may be, for example, biological,nonbiological, organic, inorganic, or a combination thereof, and may bein forms including particles, strands, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates, and slidesdepending upon the intended use.

Supports having a surface to which arrays of nucleic acids are attachedare also referred to herein as “biological chips”. The support ispreferably, silica or glass, and can have the thickness of a microscopeslide or glass cover slip. Supports that are transparent to light areuseful when the assay involves optical detection, as described, e.g., inU.S. Pat. No. 5,545,531. Other useful supports include Langmuir Blodgettfilm, germanium, (poly)tetrafluorethylene, polystyrene,(poly)vinylidenedifluoride, polycarbonate, gallium arsenide, galliumphosphide, silicon oxide, silicon nitride, and combinations thereof. Inone embodiment, the support is a flat glass or single crystal silicasurface with relief features less than about 10 Angstroms.

The surfaces on the solid supports will usually, but not always, becomposed of the same material as the substrate. Thus, the surface maycomprise any number of materials, including polymers, plastics, resins,polysaccharides, silica or silica based materials, carbon, metals,inorganic glasses, membranes, or any of the above-listed substratematerials. Preferably, the surface will contain reactive groups, such ascarboxyl, amino, and hydroxyl. In one embodiment, the surface isoptically transparent and will have surface Si—OH functionalities suchas are found on silica surfaces. In other embodiments, the surface willbe coated with functionalized silicon compounds (see, for example, U.S.Pat. No. 5,919,523).

Surface Density

The nucleic acid arrays described herein can have any number of nucleicacid sequences selected for different applications. Typically, there maybe, for example, about 100 or more, or in some embodiments, more than10⁵ or 10⁸. In one embodiment, the surface comprises at least 100 probenucleic acids each preferably having a different sequence, each probecontained in an area of less than about 0.1 cm², or, for example,between about 1/mm² and 10,000/mm², and each probe nucleic acid having adefined sequence and location on the surface. In one embodiment, atleast 1,000 different nucleic acids are provided on the surface, whereineach nucleic acid is contained within an area less than about 10⁻³ cm²,as described, for example, in U.S. Pat. No. 5,510,270.

Arrays of nucleic acids for use in gene expression monitoring aredescribed in PCT WO 97/10365, the disclosure of which is incorporatedherein. In one embodiment, arrays of nucleic acid probes are immobilizedon a surface, wherein the array comprises more than 100 differentnucleic acids and wherein each different nucleic acid is localized in apredetermined area of the surface, and the density of the differentnucleic acids is greater than about 60 different nucleic acids per 1cm².

Arrays of nucleic acids immobilized on a surface which may be used alsoare described in detail in U.S. Pat. No. 5,744,305, the disclosure ofwhich is incorporated herein. As disclosed therein, on a substrate,nucleic acids with different sequences are immobilized each in a knownarea on a surface. For example, 10, 50, 60, 100, 10³, 10⁴, 10⁵, 10⁶,10⁷, or 10⁸ different monomer sequences may be provided on thesubstrate. The nucleic acids of a particular sequence are providedwithin a known region of a substrate, having a surface area, forexample, of about 1 cm² to 10⁻¹⁰ cm². In some embodiments, the regionshave areas of less than about 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10^(−6, 10)⁻⁷, 10⁻⁸, 10⁻⁹, or 10⁻¹⁰ cm². For example, in one embodiment, there isprovided a planar, non-porous support having at least a first surface,and a plurality of different nucleic acids attached to the first surfaceat a density exceeding about 400 different nucleic acids/cm², whereineach of the different nucleic acids is attached to the surface of thesolid support in a different known region, has a different determinablesequence, and is, for example, at least 4 nucleotides in length. Thenucleic acids may be, for example, about 4 to 20 nucleotides in length.The number of different nucleic acids may be, for example, 1000 or more.In the embodiment where polynucleotides of a known chemical sequence aresynthesized at known locations on a substrate, and binding of acomplementary nucleotide is detected, and wherein a fluorescent label isdetected, detection may be implemented by directing light to relativelysmall and precisely known locations on the substrate. For example, thesubstrate is placed in a microscope detection apparatus foridentification of locations where binding takes place. The microscopedetection apparatus includes a monochromatic or polychromatic lightsource for directing light at the substrate, means for detectingfluoresced light from the substrate, and means for determining alocation of the fluoresced light. The means for detecting lightfluoresced on the substrate may in some embodiments include a photoncounter. The means for determining a location of the fluoresced lightmay include an x/y translation table for the substrate. Translation ofthe substrate and data collection are recorded and managed by anappropriately programmed digital computer, as described in U.S. Pat. No.5,510,270.

Applications Using Nucleic Acid Arrays

The methods and compositions described herein may be used in a range ofapplications including biomedical and genetic research as well asclinical diagnostics. Arrays of polymers such as nucleic acids may bescreened for specific binding to a target, such as a complementarynucleotide, for example, in screening studies for determination ofbinding affinity and in diagnostic assays. In one embodiment, sequencingof polynucleotides can be conducted, as disclosed in U.S. Pat. No.5,547,839. The nucleic acid arrays may be used in many otherapplications including detection of genetic diseases such as cysticfibrosis, diabetes, and acquired diseases such as cancer, as disclosedin U.S. patent application Ser. No. 08/143,312. Genetic mutations may bedetected by sequencing by hydridization. In one embodiment, geneticmarkers may be sequenced and mapped using Type-IIs restrictionendonucleases as disclosed in U.S. Pat. No. 5,710,000.

Other applications include chip based genotyping, species identificationand phenotypic characterization, as described in U.S. patent applicationSer. No. 08/797,812, filed Feb. 7, 1997, and U.S. application Ser. No.08/629,031, filed Apr. 8, 1996. Still other applications are describedin U.S. Pat. No. 5,800,992.

Gene expression may be monitored by hybridization of large numbers ofmRNAs in parallel using high density arrays of nucleic acids in cells,such as in microorganisms such as yeast, as described in Lockhart etal., Nature Biotechnology, 14:1675-1680 (1996). Bacterial transcriptimaging by hybridization of total RNA to nucleic acid arrays may beconducted as described in Saizieu et al., Nature Biotechnology, 16:45-48(1998). Accessing genetic information using high density DNA arrays isfurther described in Chee, Science 274:610-614 (1996).

Still other methods for screening target molecules for specific bindingto arrays of polymers, such as nucleic acids, immobilized on a solidsubstrate, are disclosed, for example, in U.S. Pat. No. 5,510,270. Thefabrication of arrays of polymers, such as nucleic acids, on a solidsubstrate, and methods of use of the arrays in different assays, arealso described in: U.S. Pat. Nos. 5,677,195, 5,624,711, 5,599,695,5,445,934, 5,451,683, 5,424,186, 5,412,087, 5,405,783, 5,384,261,5,252,743 and 5,143,854; PCT WO 92/10092; and U.S. application Ser. No.08/388,321, filed Feb. 14, 1995.

Devices for concurrently processing multiple biological chip assays areuseful for each of the applications described above (see, for example,U.S. Pat. No. 5,545,531). Methods and systems for detecting a labeledmarker on a sample on a solid support, wherein the labeled materialemits radiation at a wavelength that is different from the excitationwavelength, which radiation is collected by collection optics and imagedonto a detector which generates an image of the sample, are disclosed inU.S. Pat. No. 5,578,832. These methods permit a highly sensitive andresolved image to be obtained at high speed. Methods and apparatus fordetection of fluorescently labeled materials are further described inU.S. Pat. Nos. 5,631,734 and 5,324,633.

Preferred Embodiments

In view of the technologies provided above, the present inventionprovides in one preferred embodiment, a method of preparing a nucleicacid array on a support, wherein each nucleic acid occupies a separateknown region of the support and the nucleic acids are synthesized usingthe steps:

(a) activating a region of the support;

(b) attaching a nucleotide to a first region, the nucleotide having amasked reactive site linked to a protecting group;

(c) repeating steps (a) and (b) on other regions of the support wherebyeach of the other regions has bound thereto another nucleotidecomprising a masked reactive site link to a protecting group, whereinthe another nucleotide may be the same or different from that used instep (b);

(d) removing the protecting group from one of the nucleotides bound toone of the regions of the support to provide a region bearing anucleotide having an unmasked reactive site;

(e) binding an additional nucleotide to the nucleotide with an unmaskedreactive site;

(f) repeating steps (d) and (e) on regions of the support until adesired plurality of nucleic acids is synthesized, each nucleic acidoccupying separate known regions of the support;

wherein the surface of the substrate is maintained in a position whichis vertical or within about 30 degrees of vertical during the attachingand binding steps, and

wherein the substrate is rotated around an axis perpendicular to thesurface by an amount of from about 20 degrees to about 180 degrees, therotating being done prior to, coincident with or subsequent to at leastone of the attaching or binding steps.

Preferably, the “activating” of step (a) is carried out using a channelblock or photolithography technique, more preferably a photolithographytechnique. The “attaching” of step (b) is typically carried out usingchemical means to provide a covalent bond between the nucleotide and asurface functional group present in the first region. In someembodiments, the surface functional group will be a group present on anucleotide or nucleic acid that is already attached to the solidsupport. For example, nucleic acid arrays can be prepared using a solidsupport having a surface coated with poly-A nucleic acids to providesuitable spacing between the surface of the support and the nucleicacids that will be used in subsequent hybridization assays. Accordingly,the “attaching” can be, for example, by formation of a covalent bondbetween surface Si—OH groups and a group present on the first nucleotideof a nascent nucleic acid chain, or by formation of a covalent bondbetween groups present in a support-bound nucleic acid and a grouppresent on the first nucleotide of a nascent nucleic acid. Typically,the groups present on nucleic acids which are used in covalent bondformation are the 3′- or 5-hydroxyl groups in the sugar portion or themolecule, or phosphate groups attached thereto.

The nucleotides used in this and other aspects of the present inventionwill typically be the naturally-occurring nucleotides, derived from, forexample, adenosine, guanosine, uridine, cytidine and thymidine. Incertain embodiments, however, nucleotide analogs or derivatives will beused (e.g., those nucleosides or nucleotides having protecting groups oneither the base portion or sugar portion of the molecule, or havingattached or incorporated labels, or isosteric replacements which resultin monomers that behave in either a synthetic or physiologicalenvironment in a manner similar to the parent monomer). The nucleotideswill typically have a protecting group which is linked to, and masks, areactive group on the nucleotide. A variety of protecting groups areuseful in the invention and can be selected depending on the synthesistechniques employed. For example, channel block methods can use acid- orbase-cleavable protecting groups to mask a hydroxyl group in anucleotide. After the nucleotide is attached to the support or growingnucleic acid, the protecting group can be removed by flowing an acid orbase solution through an appropriate channel on the support.

Similarly, photolithography techniques can use photoremovable protectinggroups. Some classes of photoremovable protecting groups include6-nitroveratryl (NV), 6-nitropiperonyl (NP), methyl-6-nitroveratryl(MeNV), methyl-6-nitropiperonyl (MeNP), and 1-pyrenylmethyl (PyR), whichare used for protecting the carboxyl terminus of an amino acid or thehydroxyl group of a nucleotide, for example. 6-nitroveratryloxycarbonyl(NVOC), 6-nitropiperonyloxycarbonyl (NPOC),methyl-6-nitroveratryloxycarbonyl (MeNVOC),methyl-6-nitropiperonyloxycarbonyl (MeNPOC), 1-pyrenylmethyloxycarbonyl(PyROC), which are used to protect the amino terminus of an amino acidare also preferred. Clearly, many photosensitive protecting groups aresuitable for use in the present invention (see, U.S. Pat. No. 5,489,678and PCT WO 94/10128).

Additional photoremovable protecting groups such as5′-O-pyrenylmethyloxy carbonyl (PYMOC) andmethylnitropiperonyloxycarbonyl (MeNPOC) have been described in thecopending U.S. patent application Ser. No. 08/630,148, filed Apr. 10,1996, the contents of which are hereby incorporated by reference.

In addition to the above-described protecting groups, the presentinvention employs protecting groups, such as the 5′-X-2′-deoxythymidine2-cyanoethyl 3′-N,N-diisopropylphosphoramidites in various solvents. Inthese protecting groups, X may represent the following photolabilegroups: ((α-methyl-2-nitropiperonyl)-oxy)carbonyl (MeNPOC),((Phenacyl)-oxy)carbonyl (PAOC), O-(9-phenylxanthen-9-yl) (PIXYL), and((2-methylene-9,10-anthraquinone)-oxy)carbonyl (MAQOC).

Various methods for generating protected monomers have been described bythe U.S. Pat. No. 5,744,305, which is incorporated by reference.Detailed methods for using photoremovable protecting groups aredescribed in the U.S. Pat. No. 5,424,186, which is also herebyincorporated by reference.

The removal rate of the protecting groups depends on the wavelength andintensity of the incident radiation, as well as the physical andchemical properties of the protecting group itself. Preferred protectinggroups are removed at a faster rate and with a lower intensity ofradiation. For example, at a given set of conditions, MeNVOC and MeNPOCare photolytically removed faster than their unsubstituted parentcompounds, NVOC and NPOC, respectively.

In addition to the above-described references, photocleavable protectinggroups and methods of using such photocleavable protecting groups forpolymer synthesis have been described in the copending application Ser.No. 08/630,148 (filed Apr. 10, 1996) and Ser. No. 08/812,005 (filed Mar.5, 1997) which are incorporated by reference herein.

Step (c) provides that steps (a) and (b) can be repeated to attachnucleotides to other regions of the solid support.

One of skill in the art will appreciate that steps (a) and (b) can berepeated a number of times to produce a solid support having a layer ofattached nucleotides. Preferably, each attached nucleotide is in a knownposition.

In subsequent steps (d), (e) and (f), the protecting group is removedfrom one of the nucleotides to reveal a reactive site on the nucleotide.Thereafter, an additional nucleotide (optionally having a maskedreactive site attached to a protecting group) is attached to thesupport-bound nucleotide. As above, these steps can be repeated toselectively attach or bind an additional nucleotide to any of thesupport-bound nucleotides. Still further, the steps of deprotecting andattaching an additional nucleotide can be carried out on the newly addednucleotides to continue the synthesis of the nascent nucleic acid.

As noted above, the above steps are preferably carried out incombination with substrate rotation. In one group of embodiments, awafer (or solid support) is divided into 49 synthesis zones (chips) andplaced in a substantially horizontal position in an irradiation chamber.Portions of the chips are illuminated through a photolithography mask toactivate known regions of the chips. The wafer is then transferred to asynthesis apparatus wherein the wafer is held in a substantiallyvertical position and a flowcell having certain reagents is attached tothe wafer. For wafers that are square, the position is shown in FIG. 1(having a vertex pointing down). After the wafer is contacted with thedesired reagents and attaching or binding has taken place, excessreagents can be removed by washing and the wafer can be transferred tothe irradiation (or activation) chamber. Again, known regions of thewafer are illuminated to activate those regions. The wafer is againtransferred to the synthesis apparatus and rotated prior to contactingthe wafer with the desired reagents. The process can be continued untilan array having a desired number of attached oligonucleotides has beenprepared.

In a further preferred embodiment, the preparing comprises:

a) removing a photoremovable protecting group from at least a first areaof a surface of a substrate, the substrate comprising immobilizednucleotides on the surface, and the nucleotides capped with aphotoremovable protective group, without removing a photoremovableprotecting group from at least a second area of the surface;

b) simultaneously contacting the first area and the second area of thesurface with a first nucleotide to couple the first nucleotide to theimmobilized nucleotides in the first area, and not in the second area,the first nucleotide capped with a photoremovable protective group;

c) removing a photoremovable protecting group from at least a part ofthe first area of the surface and at least a part of the second area;

d) simultaneously contacting the first area and the second area of thesurface with a second nucleotide to couple the second nucleotide to theimmobilized nucleotides in at least a part of the first area and atleast a part of the second area;

e) performing additional removing and nucleotide contacting and couplingsteps so that a matrix array of at least 100 nucleic acids havingdifferent sequences is formed on the support;

wherein the surface of said substrate is maintained in a position whichis vertical or within about 30 degrees of vertical, and

wherein the substrate is rotated around an axis perpendicular to saidsurface by an amount of from about 20 degrees to about 180 degrees, saidrotating being done prior to, coincident with or subsequent to at leastone of said attaching or binding steps.

In this embodiment of the invention, the steps of removingphotoremovable protecting groups, coupling nucleotides to specificareas, removing protecting groups from the coupled nucleotides, andcoupling additional nucleotides can all be carried out as described in,for example, U.S. Pat. No. 5,510,270, with the added feature that thesubstrate is rotated during the course of synthesis.

In still further preferred embodiments, the nucleoside phosphoramiditemonomers used in the methods described above have the formula:

wherein B represents adenine, guanine, thymine, cytosine, uracil oranalogs thereof; R is hydrogen, hydroxy, protected hydroxy, halogen oralkoxy; P is a phosphoramidite group; and PG is a photoremovableprotected group.

In the group of embodiments using monomers of formula (I), B ispreferably adenine, guanine, thymine, cytosine or uracil. Morepreferably, B is adenine, guanine, thymine, or cytosine, and R ishydrogen. Still more preferably, the array prepared using the monomersabove comprises at least 10 different nucleic acids, more preferably atleast 100 different nucleic acids, still more preferably at least 1000different nucleic acids. Most preferably, the array comprises at least10,000 to 100,000 or more different nucleic acids. Additionally, eachdifferent nucleic acid is in a region having an area of less than about1 cm², more preferably less than about 1 mm².

In still other preferred embodiments, B is adenine, guanine, thymine, orcytosine; R is hydrogen.

In further preferred embodiments, B is adenine, guanine, thymine, orcytosine; R is hydrogen; and PG is MeNPOC.

In still further preferred embodiments, B is adenine, guanine, thymine,or cytosine; R is hydrogen; PG is MeNPOC, and P is —P(OCH₂CH₂CN)N(iPr)₂.

One of skill in the art will appreciate that the present invention canbe readily modified to use protected nucleoside phospohoramiditemonomers wherein the protecting group on the 5′ hydroxy is acid or baseremovable. Such modifications will render the invention applicable toother synthesis methodologies such as flow channel and spotting methodsdescribed in more detail above. Regardless of the array synthesismethods, substrate rotation provides an enhancement in both quantity andperformance of the resulting chips or arrays.

EXAMPLES Example 1

This example illustrates the improvement in oligonucleotide arrayconstruction and performance that can be achieved using the methodsdescribed herein.

Test vehicles, checkerboard of the “213” and “Block 3” sequences wereused to perform preliminary experiments. For each of the Checkerboardand Control Block 3 arrays, the oligonucleotides were synthesized in a23-step process to form arrays of 20-mers. Fluorescein stain, 0.5 hourat 35° C. hybridization assay was used to indicate the variation betweenarrays synthesized. The results were then confirmed by synthesizing Geneexpression arrays (e.g., Mu11K), a 74-step process. The standard QCassay, Phycoerythrin/streptavidan (SAPE) stain, 18 hours at 45° C.hybridization assay, was used to evaluate the arrays. The averagedifference of the “Block3” and “Bio C” were analyzed on the GeneExpression probe arrays.

In each step, a square wafer is placed in an illumination chamber andlight is directed at the wafer through a mask to selectively deprotectand activate specific sites on the wafers. The wafers are thentransferred to a synthesis chamber wherein the wafers are held in avertical position with one corner of the square wafer pointing down. Aflowcell is contacted with the wafer and reagents are directed onto thewafer from the bottom to the top. Prior to each step in the synthesischamber, the wafers are rotated in multiples of 90° (e.g, 90°, 180°,270°) so that one corner of the wafer is always pointed downward. TABLE1 Number Coefficient Top to of of Variation Bottom Rotation Type ofArray wafers (%) Ratio type Checkerboard 213 - control 4 17%, 21%, 1.4,1.5, 19%, 20% 1.4, 1.4 Checkerboard 213 - rotated 2 6%, 6% 1.0, 1.0 90degrees Checkerboard Block 3 - 1  8% 1.4 control Checkerboard Block 3 -1  2% 1.0 90 degrees rotated Murine gene expression - 2 55%, 77% 2.9,5.0 Block 3 Control Murine gene expression - 1 19% 1.2 random 90 Block 3Rotated degrees Murine gene expression - 2 68%, 78% 3.7, 4.9 BioC-Control Murine gene expression - 1 35% 1.7 random 90 Bio C - Rotateddegrees

As can be seen from the table of data above, rotating the wafers duringsynthesis led to a significant reduction in intra wafer variability offrom about 50% (68% CV versus 35% CV) to about 75% (8% CV versus 2% CV).

FIG. 2 is a graph that illustrates the average difference in controlversus rotated arrays. In this experiment, lot 95296 as a controlexhibited Block 3 average differences of about 50-150. The rotated lots,95297 and 95299, exhibited average differences of about 45-85 and about40-70, respectively, indicating the reduced variability for rotatedlots.

FIG. 3 provides an analysis of chips from different portions of eachwafer. In this graph, b=bottom, c=center, t=top, l=left and r=right. Ascan be seen in FIG. 3, the control wafer (296) exhibited markedly widerranges of average difference, while the rotated wafers (297 and 299)provided more consistent results between the portions of the wafer.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of preparing a nucleic acid array on a support, wherein eachnucleic acid occupies a separate known region of the support, saidsynthesizing comprising: (a) activating a region of the support; (b)attaching a nucleotide to a first region, said nucleotide having amasked reactive site linked to a protecting group; (c) repeating steps(a) and (b) on other regions of said support whereby each of said otherregions has bound thereto another nucleotide comprising a maskedreactive site link to a protecting group, wherein said anothernucleotide may be the same or different from that used in step (b); (d)removing the protecting group from one of the nucleotides bound to oneof the regions of the support to provide a region bearing a nucleotidehaving an unmasked reactive site; (e) binding an additional nucleotideto the nucleotide with an unmasked reactive site; (f) repeating steps(d) and (e) on regions of the support until a desired plurality ofnucleic acids is synthesized, each nucleic acid occupying separate knownregions of the support; wherein the surface of said substrate ismaintained in a position which is vertical or within about 30 degrees ofvertical, and wherein the substrate is rotated around an axisperpendicular to said surface by an amount of from about 20 degrees toabout 180 degrees, said rotating being done prior to, coincident with orsubsequent to at least one of said attaching or binding steps.
 2. Amethod in accordance with claim 1, wherein said rotating is conductedprior to, coincident with or subsequent to at least 50% of saidattaching or binding steps.
 3. A method in accordance with claim 1,wherein said rotating is conducted prior to, coincident with orsubsequent to at least 80% of said attaching or binding steps.
 4. Amethod in accordance with claim 1, wherein said rotating is in an amountof from about 75 to about 105 degrees.
 5. A method in accordance withclaim 1, wherein said rotating is in an amount of about 90 degrees.
 6. Amethod in accordance with claim 1, wherein said interface is vertical orwithin about 10 degrees of vertical and said rotating is in an amount ofabout 90 degrees.
 7. A method in accordance with claim 1, wherein saidsubstrate is a substantially square planar silica chip, said interfaceis vertical or within about 10 degrees of vertical and said rotating isin an amount of about 90 degrees.
 8. A method in accordance with claim7, wherein said substantially square planar silica chip is held in avertical position with one of the four square verticies pointingdownward.
 9. A method in accordance with claim 1, wherein at least 10different nucleic acids are formed on said surface.
 10. A method inaccordance with claim 1, wherein at least 100 different nucleic acidsare formed on said surface.
 11. A method in accordance with claim 1,wherein at least 1000 different nucleic acids are formed on saidsurface.
 12. A method in accordance with claim 1, wherein at least10,000 different nucleic acids are formed on said surface.
 13. A methodin accordance with claim 1, wherein at least 100,000 different nucleicacids are formed on said surface.
 14. A method in accordance with claim1, wherein each different nucleic acid is in a region having an area ofless than about 1 cm².
 15. A method in accordance with claim 1, whereineach different nucleic acid is in a region having an area of less thanabout 1 mm².